Angiogenesis is a tightly regulated process in various aspects, in the mature mammalian organisms, the physiological angiogenesis only occurs in ovary, uterus and placenta, whereas in other tissues, upon angiogenesis, it will be maintained in a high degree of stability, and regulated by many positive and negative regulatory factors, thereby maintaining a homeostasis state; while the pathological angiogenesis involves in a series of disease processes, such as wound healing, malignant tumor, diabetic retinopathy, rheumatoid arthritis and so on (Peter B, et al. Curr Opin Genet Dev, 2005, 15: 102-111).
In the early 1970s, Judah Folkman first proposed that the growth and migration of the malignant tumor are both closely related to the tumor angiogenesis, upon growing up to a diameter of 2˜3 mm, a majority of tumors need new blood vessels to supply nutrients and oxygen (Folkman J. N Engl J Med, 1971, 285: 1182-1186); as compared with those in the tumor tissues, the vascular endothelial cells in the normal tissues are in a resting state, and the blood vessels are mature and stable; and the vessel cells in the tumor tissues have the following characteristics, such as rapid proliferation, irregular vascular grading, chaotic network, etc. (Dietmar W, et al. Cancer Treat Rev, 2011, 37: 63-74). Therefore, the vascular system of the tumor becomes a very important therapeutic target. At present, in most of the literatures, the vascular targeting agents tend to be divided into angiogenesis inhibitors (anti-angiogenic drugs or angiogenesis inhibitors (AIs) and vascular disrupting agents (VDAs) (Patherson D M, et al. Clin Oncol (R Coll Radiol), 2007, 19: 443-456). AIs inhibit the tumor neovascularization mainly by inhibiting the matrix degradation, inhibiting the activation of the angiogenic factors and affecting the proliferation of the tumor vascular endothelial cells, etc. (Folkman J. Nat Rev Drug Discov. 2007, 6: 273-286). Numerous clinical data have confirmed that, AIs can effectively inhibit the tumor progression, reduce the occurance of tumor metastasis by inhibiting angiogenesis (Gasparini G, et al. Nat Clin Pract Oncol. 2005, 2: 562-577); and VDAs can quickly and widely disrupt the formed tumor blood vessels, resulting in a large area of tumor necrosis due to internal ischemia, thereby inhibiting the tumor growth (Philip E T. Clin Cancer Res, 2004, 10: 415-427). Therefore, inhibition of tumor angiogenesis and disruption of the formed tumor blood vessels have become effective strategies in developing antitumor drugs.
Diabetic retinopathy is a serious complication of diabetes, which seriously affects the quality of life of the diabetic patient. (Malone J I, et al. Diabetes Care, 2001, 24: 522-526; Ramavat P R, et al. J Clin Diagn Res, 2013, 7: 1387-1390). Although the exact pathogenesis of diabetic retinopathy is not completely clear at present, some studies have shown that the retinal neovascularization involved in the disease process (Jae S Y, et al. Diabetes Metab J, 2013, 37: 262-269). In recent years, it has been found in clinic that bevacizumab, as a receptor antagonist of VEGF (vascular endothelial growth factor), can be used to treat the retinal angiogenesis of the diabetic retinopathic patients, and has achieved a certain effect (Zhao L Q, et al. Br J Ophthalmol, 2011, 95: 1216-1222). However, VEGF receptor antagonists can not completely suppress the retinal angiogenesis of the diabetic retinopathic patient (Watanabe D, et al. N Engl J Med, 2005, 353: 782-792). Thus, it is of great significance to find more effective drugs for inhibiting the diabetic retinopathy.
On the other hand, rheumatoid arthritis is an autoimmune disease, a refractory disease characterized by eroding cartilage in combination with chronic synovitis, and resulting in the destruction of bone and joint. The pathogenesis of rheumatoid arthritis has not yet been completely clarified hitherto, and there are no specific therapeutics for the same. It has been reported that synovial angiogenesis accompanying with inflammatory cell infiltration is an important pathological feature for the pannus formation in rheumatoid arthritis and the destruction in joint (Lioté F. Rev Prat, 1993, 43: 2239-2245; Roccaro A M, et al. Curr Drug Targets Inflamm Allergy, 2005, 4: 27-30; Hirohats S, et al. Lancet, 1999, 353:1331-1334). Inhibition of the synovial angiogenesis of the rheumatoid arthritis patient has currently become one of the effective strategies for treating rheumatoid arthritis.
Vinca alkaloids comprise vinblastine and vincristine separated from Apocynaceae Catharanthus roseus, and their derivatives vinorelbine and vinflunine, all of them belonging to bisindole alkaloids. Vinblastine (VLB) and vincristine (VCR) are natural original bisindole alkaloids (Beer M T. Br Emp Cancer Campaign, 1955, 33: 487-489; Gorman M, et al. J Am Chem Soc, 1959, 81: 4745-4746). Pharmacological studies shown that, vinblastine and analogues or derivatives thereof are cytotoxic drugs, which mainly inhibit the polymerization of tubulins, hinder the formation of spindle microtubules and arrest the cell nucleus division in metaphase (Olmsted J B, et al. Annu Rev Biochem, 1973, 42: 507-509). Vinblastine and the derivatives thereof have broad-spectrum anticancer activities, and are mainly used to treat Hodgkin's disease and chorionic epithelioma in clinic, and have some therapeutic effects on acute leukemia, breast cancer, ovarian cancer, testicular cancer, head and neck cancer, oropharyngeal cancer, and monocytic leukemia (Wilson L. Ann NY Acad Sci USA, 1975, 253: 213-214). In recent years, Angelo Vacca et al have found that vinblastine at a non-toxic dose can significantly inhibit neovasculature at cell levels (Angelo V, et al. Blood, 1999, 94: 4143-4155); the studies of Giannoula Klement et al have shown that the tumor neovascularization can be inhibited by continuous supplying a low dose of vinblastine (Giannoula K, et al. J Clin Invest, 2000, 105: R15-24); James Moore, et al have confirmed that vincristine can inhibit the growth of the new tumor blood vessels (James M, et al. J Pediatr Surg, 2001, 36: 1273-1276); on the other hand, Anna Kruczynskia et al have found that vinflunine can inhibit tumor angiogenesis, and at the same time disrupt the formed tumor blood vessels, and also have significant inhibitions on the experimental malignant tumor metastasis (Anna K, et al. Eur J Cancer, 2006, 42: 2821-2832). However, the applications and studies of vinca alkaloids on diabetic retinopathy, rheumatoid arthritis, etc., have not been reported yet.
Like many chemotherapy drugs used in clinic, vinca alkaloids have many serious side effects during the treatment of diseases, such as bone marrow suppression, myalgia, nausea and vomiting, and other adverse effects (Magnus P, et al. J Am Chem Soc, 1987, 109: 7929-7930), which greatly limit their applications in clinical practice. One of the effective ways to reduce the toxic side effects of such drugs is to make structural modifications on the drugs to produce prodrugs, and make the prodrugs selectively acting on the genes, enzymes, signal transduction factors, etc., of the lesion target cells, by virtue of the molecular biological differences between the lesion tissues and the normal tissues, so as to achieve the purpose of targeted therapy. In recent years, a large number of studies have shown that fibroblast-activation protein α (FAPα) is specifically expressed in the callus tissues and on the surfaces of more than 90% of the tumor tissue activated fibroblasts and pericytes (Teresa R M, et al. Oncogene, 2004, 23: 5435-5446), on the surfaces of osteoarthritis chondrocytes (Jennifer M M, et al. Arthritis Res Ther, 2006, 8: R23), and also significantly highly expressed on the surfaces of myofibroblasts in the vitreous retinopathic tissues (Jennifer M M, et al. Acta Ophthalmol, 2011, 89: 115-121). In summary, in the present invention, vinca alkaloids are chemically modified to form FAPα enzyme-activated prodrugs, so as to achieve the purposes of targeting, reducing toxiciy and increasing efficiency.
Contents of the Invention
In order to achieve the targeting property of vinca alkaloid drugs, overcome the serious toxic side effects of such drugs in the prior art, and improve the therapeutic effects of the drugs, the present invention provides a new kind of vinca alkaloid derivatives (including hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives) and physiologically acceptable salts thereof, the particular technical solutions of the invention are as follows:
The present invention provides a kind of vinca alkaloid dipeptide derivatives or their physiologically acceptable salt thereof, wherein the vinca alkaloid dipeptide derivatives are selected from the structures shown as follows, designated as BX-CCXJ, BX-CCJ, BX-CCRB and BX-CCFN, respectively,
wherein, Z-GP- represents a benzyloxycarbonyl glycyl-prolyl group having the following structure,
The present invention also provides a method for preparing the vinca alkaloid dipeptide derivatives mentioned above, wherein the synthetic route is as follows

specifically comprising the following steps:
S1. The vinca alkaloids or their salt thereof are dissolved in an organic solvent, added hydrazinolyzed hydrate, heated with stirring and reacted in dark under nitrogen protection for 10˜60 hours; with the reaction temperature controlled at 40° C.˜120° C.; after completion of the reaction, separated and purified, thus obtaining the hydrazinolyzed vinca alkaloids (R—NHNH2);
S2. the hydrazinolyzed vinca alkaloids (R—NHNH2), benzyloxycarbonyl glycyl-proline (Z-GP-OH) and a condensing agent, are stirred and reacted in dark at −10° C.˜50° C.; after completion of the reaction, quenched with water, separated and purified, thus obtaining the vinca alkaloid dipeptide derivatives (Z-GP-NHNH—R).
As a preferred embodiment, the hydrazinolyzed hydrate in S1 is 40 wt %˜80 wt % of hydrazinolyzed hydrate; the molar feeding ratio between the vinca alkaloids and the hydrazinolyzed hydrate is 1:5˜1200. The organic solvent in S1 is methanol.
As a preferred embodiment, the condensing agent in S2 is selected from one of or a mixture of more of ethyl chloroformate, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), N,N′-diisopropyl carbodiimide (DIC), benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (PyBOP) and 1-chloro-N,N′,2-trimethylacrylamide.
As a preferred embodiment, the molar feeding ratio among the hydrazinolyzed vinca alkaloids, benzyloxycarbonyl glycyl proline (Z-GP-OH) and the condensing agent in S2 is 1:1.05˜3.0:1.05˜3.0.
Among them, the vinca alkaloids are selected from vincristine, vinblastine, vinflunine, vinorelbine or their salt thereof; the hydrazinolyzed vinca alkaloids are selected from JJ-CCXJ, JJ-CCJ, JJ-CCRB and JJ-CCFN shown as follows.

Another object of the present invention is to provide a kind of hydrazinolyzed vinca alkaloids or their physiologically acceptable salt thereof, wherein the hydrazinolyzed vinca alkaloids are selected from JJ-CCXJ, JJ-CCJ, JJ-CCRB and JJ-CCFN as shown above.
The present invention also provides a kind of physiologically acceptable salt of the vinca alkaloid dipeptide derivatives formed from the above mentioned vinca alkaloid dipeptide derivatives and a method for preparing the same.
As a preferred embodiment, the physiologically acceptable salt of the vinca alkaloid-dipeptide derivatives are selected from any one in Table 1:
TABLE 1The salt form of the vinca alkaloid dipeptide derivativesBX-CCJ•sulfateBX-CCXJ•sulfateBX-CCRB•tartrateBX-CCFN•tartrate
The vinca alkaloid dipeptide derivatives or their physiologically acceptable salt thereof described in the present invention can be present in free state in medical application.
The method for preparing the vinca alkaloid dipeptide derivatives or their physiologically acceptable salt thereof described above, are characterized in that vinca alkaloid dipeptide derivatives are dissolved in an organic solvent containing 1.05˜3.0 moles of acid (HA), stirred and reacted at −10° C.˜40° C. for 3˜20 hours, separated the solid compound, washed, then redissolved the solid compound in water, freeze-dried, thus obtaining the final products.
As a preferred embodiment, the acids are hydrochloric acid, sulfuric acid, acetic acid, tartaric acid or citric acid, and the organic solvent is a solution of methanol and dichloromethane with a volume ratio of 1:1.
The present invention also provides the application of the above mentioned hydrazinolyzed vinca alkaloids, vinca alkaloid dipeptide derivatives or their physiologically acceptable salt thereof in the preparation of the antitumor drugs, preferably, the tumor are stomach cancer, lung cancer, nasopharyngeal cancer, breast cancer, intestinal cancer, liver cancer, leukemia, lymphoma, prostate cancer, cervical cancer, melanoma, ovarian cancer, neuroblastoma, nasopharyngeal carcinoma, nephroblastoma or multidrug resistant tumor.
The present invention also provides the application of the above mentioned hydrazinolyzed vinca alkaloids, vinca alkaloid dipeptide derivatives or their physiologically acceptable salt thereof in the preparation of a medicament in the prevention or treatment of diabetic retinopathy or rheumatoid arthritis.
The present invention also provides the application of the above mentioned hydrazinolyzed vinca alkaloids, vinca alkaloid dipeptide derivatives or their physiologically acceptable salt thereof in the preparation of a medicament in serving as angiogenesis inhibitors or vascular disrupting agents.
The present invention also provides a pharmaceutical composition, comprising the above mentioned vinca alkaloid dipeptide derivatives or their physiologically acceptable salt thereof or the above mentioned hydrazinolyzed vinca alkaloids or their physiologically acceptable salt thereof.
Among them, the above mentioned physiologically acceptable salt can be selected from hydrochloride, sulfate, acetate, tartrate or citrate.
In the above mentioned applications, preferably, the vinca alkaloid dipeptide derivative can be used as a substrate for FAPα-specific hydrolysis.
The present invention has the following beneficial effects as compared with those in the prior art:
(1) The vinca alkaloid dipeptide derivatives described in the present invention can significantly reduce the toxicity in normal cells and the toxicity in vivo, and can be specifically hydrolyzed in vivo and in intro by a specific enzyme, FAPα, which cleaves the dipeptide moiety (Z-GP) and releases hydrazinolyzed vinca alkaloids.
(2) The vinca alkaloid derivatives can significantly inhibit the proliferation of a variety of tumor cell lines in vitro and the tumor growth of tumor-bearing nude mice in vivo.
(3) The vinca alkaloid derivatives of the present invention also have significant effects on inhibiting the angiogenesis and disrupting the formed new blood vessels.
(4) The vinca alkaloid derivatives of the present invention have good inhibitory effects on the invasive capacity, migration capacity and tube formation of HUVECs; and have good inhibitory effects on the angiogenesis of corneal micropocket, the angiogenesis of synovial vessels and the angiogenesis of Matrigel Plug, etc. At the same time, they have disruptive effects on the formed HUVEC tubes, corneal micropocket vessels, synovial vessels and Matrigel Plug vessels.
(5) The in vivo and in vitro drug efficacy tests have shown that the vinca alkaloid derivatives of the present invention can be applied in the treatment and prevention of the diseases, such as malignant tumor, diabetic retinopathy, rheumatoid Arthritis, etc. Particularly, it has been also found in the present invention that the applications of the vinca alkaloid derivatives on the diseases, such as malignant tumor, diabetic retinopathy, rheumatoid arthritis, etc., have better drug efficacies, as compared with those of the vinca alkaloids, and there are significant differences between them in drug efficacy.
(6) The synthetic methods of the present invention have the characteristics of mild conditions, simple procedure, high yield, high purity, good economy and practicality, etc. Particularly, the hydrazinolyzed vinca alkaloids of the present invention can be used as the synthetic intermediates of the vinca alkaloid dipeptide derivatives, moreover they also have good physiological activities, and can be applied in the preparation of the related drugs as the active ingredients.
In FIGS. 3-16, *refers to P<0.05, **refers to P<0.01